It has been well established that post-translational modifications by the Small Ubiquitin-like Modifier (SUMO) family of proteins are critical events in DNA damage response in mammalian cells. However, the mechanism of how SUMOylation is involved in DNA repair remains largely unclear. Among the different types of DNA damage, DNA double-strand break is the most dangerous type that leads to cell death and mutations. DNA double-strand break repair is also highly relevant to radiation and chemotherapy for cancer treatment. In the proposed study, I will test a hypothesis that the SUMO-mediated protein-protein interactions are critical to the repair of DNA double-strand breaks. This hypothesis is supported by my preliminary data that inhibition of SUMO-dependent protein-protein interactions in cancer cells inhibits the repair of DNA double-strand breaks, and sensitizes tumor cells to radiation and chemotherapy. In this proposed study, a peptide containing the SUMO-binding motif (SBM), which specifically inhibits SUMO-dependent protein-protein interaction, is used to investigate the role of SUMOylation in the different pathways for the repair of DNA double-strand breaks. In mammalian cells, double-strand breaks are mostly repaired by the homologous recombination (HR) or the non-homologous end joining (NHEJ) pathways. I will examine whether SUMO-dependent protein-protein interaction is required for HR and/or NHEJ pathways using fluorescence and PCR-based assays, as well as using cell lines that are deficient in one of these repair pathways. Moreover, the SUMOylated targets that are blocked by the SBM will be identified by coimmunoprecipitation and subsequent liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. Finally, the identified proteins will be analyzed to identify their SUMOylation site and to examine their biological functions by biochemical and molecular biological approaches. The proposed studies will significantly advance our knowledge about the role of SUMOylation in DNA double-strand break repair pathways. Furthermore, results from this study will lead to the development of new therapeutic strategies to inhibit DNA repair in tumor cells and thereby reducing tumor cell resistance to radiation and chemotherapy.